Shiga toxin-producing Escherichia coli (STEC) are a group of prevalent foodborne pathogens responsible for outbreaks of human gastrointestinal disease. The morbidity and mortality associated with these outbreaks have highlighted the threat these organisms pose to public health (Karch et al., Int'l J Med Microbiol, (2005) 295:405-18; Gyles, J. Anim Sci, (2007) 85:E45-62; Manning et al., Emerg Infect Dis, (2007) 13:318-21). Most STEC outbreaks have been traced worldwide to the consumption of bacterial-contaminated food. Ruminants are the main reservoir for STEC strains and food contaminated with bovine feces has been linked to severe complications, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) (Hussein, J Anim Sci, (2007) 85: E63-72).
STEC possess a number of virulence factors, but Shiga toxins (Stxs) were considered the most critical in disease pathogenesis and are responsible for HC and HUS. Stxs are AB5 holotoxins and are comprised of one A subunit (32 kDa) and five B subunits (7.7 kDa) (Fraser et al., Nat Struct Biol, (1994) 1:59-64; Fraser et al., J Biol Chem (2004) 279:27511-17). The Stx A subunit is an enzymatically active N-glycosidase that inhibits the activity of rRNA by cleavage of an adenine base from the 28S rRNA component of the eukaryotic ribosomal 60S subunit, causing protein synthesis to cease resulting in cell death (Endo and Tsurugi, J Biol Chem, (1988) 263:8735-9). The Stx B subunit is responsible for binding to host cells through interaction with globotriaosylceramide (Gb3) or globotetraosylceramide (Gb4) receptors present on the surfaces of cells (Lingwood, Adv Lipid Res (1993) 25:189-211), leading to subsequent internalization of the toxin. There are two serologically distinct groups of Stxs, Stx1 and Stx2. Recent epidemiological and molecular typing studies suggested that STEC strains expressing Stx2 were more virulent than strains expressing either Stx1 or both Stx1 and Stx2 (Ostroff et al., J Infect Dis, (1989) 160:994-8; Boerlin et al., J Clin Microbiol, (1999) 37:497-503). A mean lethal dose (LDso) for Stx2 of 50 ng/kg in mice was reported by Tesh et al. (Infect Immun, (1993) 61:3392-402) and Lindgren et al. (Infect Immun, (2003) 69:623-31). In contrast to Stx1, many variants of Stx2 have been identified (Weinstein et al., J Bacteriol, (1988) 170:4223-30; Piérard et al., J Clin Microbiol (1998) 36:3317-22; Bertin et al., J Clin Microbiol, (2001) 39:3060-5; Leung et al., Appl Environ Microbiol, (2003) 69:7549-53; Strauch et al., Infect Immun, (1994) 40:338-43). These variants differ from each other in terms of their affinity for host receptors, cytotoxicity, and pathogenicity.
The capacity to control STEC disease in humans and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection. Although the role of Stx2 in these outbreaks has received considerable attention, rapid, sensitive and specific detection methods for this toxin in food are still limited. This is because detection of Stxs in food samples is often difficult due to the combination of low toxin concentration and effect of the complex matrix present in food. Historically, the Vero cell cytotoxicity assay has played an important role in establishing a diagnosis of STEC infection and it still remains the “gold standard” for Stx activity. However, like most activity-based assays, such as the mouse bioassays, radioactivity assays, and cell-free translation assays, the Vero cell assay is time-consuming, requires cell culture facilities, and expensive equipment that is usually not available in many laboratories. Furthermore, a subsequent antibody-based neutralization bioassay is required in order to confirm the presence of the toxin. Other assays, such as receptor-based assays are less time-consuming and enable the discrimination of different toxins, but detailed evaluation and optimization are needed to establish these methods as analytical tools (Uzawa et al., ChemBioChem, (2007) 61:3392-402).
Over the past decades, a number of immunoassays have been developed, the most common ones being the enzyme-linked immunosorbent assays (ELISA). These assays provide multiple benefits. Notably, they are simple, rapid, cost-effective, and all reagents and equipment needed are available in most laboratories. However, the sensitivity and specificity of immunoassays is largely dependent on the quality of the antibodies used. Our recent studies on detecting botulinum neurotoxin type A in milk demonstrated that simple immunoassay formats can be highly sensitive when high-affinity antibodies are incorporated (Stanker et al., J Immunol Methods, (2008) 336:1-8). While antibodies against Stx2 have been described in the scientific literature, few are commercially available. Their expense and lack of sufficient binding affinity to the native toxins make studies focused on constructing a sensitive immunoassay difficult.
Within each Stx type (Stx1 and Stx2), there are a number of subtypes which vary in sequence, specificity, and toxicity. There are 3 characterized subtypes of Stx1 (Stx1a, Stx1c, and Stx1d) and 7 subtypes of Stx2 (Stx2a, 2b, 2c, 2d, 2e, 2f, and 2g) (Paton et al., Nat. Med., (2000) 6:265-70). The subtypes of Stx1 are relatively conserved at the amino acid level, whereas those of Stx2 can be more diverse. However, the Stx2a, Stx2c, and Stx2d subtypes are very similar to each other, and these subtypes are typically associated with HUS (Fuller et al., Infect. Immun. (2011) 79:1329-37; Orth et al., Diagn. Microbiol. Infect. Dis. (2007) 59:235-42). Stx2b, Stx2e, Stx2f, and Stx2g are less commonly found in serious human disease, although Stx2e can cause edema disease in neonatal piglets (Oanh et al., Infect. Immun. (2012) 80(1):469-73). Stx2f (found mostly in avian isolates) (Schmidt et al., Appl. Environ. Microbiol. (2000) 66:1205-8) is the most unique of the Stx2 subtypes (73.9% identity to Stx2a in the A subunits), followed by Stx2b (93.3%), Stx2e (93.9%), and finally Stx2g (94.9%). Differences among the B subunits determine each subtype's receptor specificity. Stx2a, Stx2c, and Stx2d bind preferentially to Gb3Cer, while it has been reported that Stx2e prefers Gb4Cer (but can also bind Gb3Cer) (Muthing et al., Glycobiology, (2012) 22:849-62). Several amino acids in the C-terminus of the B subunit are critical for determining receptor preference. When the double mutation Q64E/K66Q is made to the Stx2e B subunit, it loses its ability to bind Gb4Cer, and has a receptor preference analogous to Stx2a (Tyrell et al., Proc Natl Acad Sci USA (1992) 89:524-8). The B subunit of Stx2f has Q64/K66 like Stx2e, and can bind both GB-LPS and Gb4-LPS, which are mimics of Gb3Cer and Gb4Cer, respectively (Skinner et al., PLoS ONE, 8/9:e76563 (September 2013)).
Most Stx2 detection kits (both PCR and immunoassays) are optimized to Stx2a, and cross-react with closely related Stx2c and Stx2d. However, many do not recognize the divergent Stx2b, Stx2e, and Stx2f subtypes. Antibodies that recognize Stx2f have been reported, but few are commercially available and they are generally sold only as components of an assay kit, making them difficult to use as research tools and very expensive. One of the primary means for detecting Stx1 and Stx2, the PREMIER EHEC KIT from Meridian Biosciences, has been reported to detect Stx2f in two studies (Schmidt et al., Appl. Environ. Microbiol. (2000) 66:1205-8; Willford et al., J. Food Protect (2009) 72:741-7) but is insensitive to Stx2f in another (Feng et al., Appl. Environ. Microbiol. (2011) 77:6699-6702). A reverse passive latex agglutination assay (VTEC-RPLA) has repeatedly been shown to recognize Stx2f, but the sensitivity of this assay to Stx2f is unknown (Denka Seiken, Japan) (Schmidt et al., supra). Monoclonal antibodies (mAbs) that react robustly and uniquely to Stx2f with use in an immunoassay for simple detection of the Stx2f subtype is therefore desired.